This applications claims benefit to German Patent Application No. 199 19 634.6 filed Apr. 30, 1999.
The invention relates to a simple continuous test for the identification of structures which favor the arrangement of aromatics to give charge-transfer complexes, such as, for example, complex phospholipid/lipid structures (bilayer, monolayer, aggregates, micelles), with the aid of synthetic fluorescence-labeled acylglycerides, and its use for determination of the activity of lipases/lipase inhibitors.
Lipases, phospholipases, and other lipolytic enzymes have great importance in the biotechnological and medical field. In certain metabolic disorders, increased lipase activity in the fatty tissue can be detected, which is held partly responsible for the pathogenesis of this disease. The greatest part of the energy reserves of the body is stored in cells of the fatty tissue as fatty acids of the triglycerides. The essential anabolic processes caused by insulin include the stimulation of the uptake of substrates for triglyceride synthesis and the increase in lipogenesis. A further important process caused by insulin is the inhibition of lipolysis, the process by means of which catabolic hormones, primarily catecholamines, stimulates the hydrolysis of triglycerides and thereby induce the release of fatty acids. An important problem which is linked with noninsulin-dependent diabetes mellitus (NIDDM) has its cause in the uninhibited lipolysis of the fat cells, which leads to increased levels of unesterified fatty acids in the plasma. According to a present idea, the fatty acids stimulate gluconeogenesis in the liver and decrease the glucose utilization in the skeletal muscle by means of still poorly characterized molecular mechanisms. In fact, it was possible to show that the suppression of lipolysis in fat cells by inhibitors of lipolysis, such as agonists of the nicotinic acid receptor of the fat cell, lowers both the fatty acid concentrations in the plasma and raised blood sugar in diabetic animals and patients. Unfortunately, these beneficial effects are not particularly strongly pronounced and only of relatively short duration. This may be based on a physiological counterregulation caused by intervention in the regulatory mechanism of the rate-determining enzyme of lipolysis, the hormone-sensitive lipase (HSL). There are good reasons to assume that the inhibition of the lipolytic reaction will lead to an improved therapy of NIDDM, at least with respect to the suppression of the fatty acid release from the fat cells. The direct inhibition of HSL by suitable inhibitors should in this case get around the obvious difficulties of an intervention into the complex regulation of HSL.
The activity of lipolytic enzymes is traditionally investigated using radiometric, titrimetric, enzymatic, or fluorimetric/photometric methods. Radiometric assays are the most sensitive, but they require expensive radiolabeled substrates, are discontinuous, and require the separation of the radiolabeled substrate from the radiolabeled product. Such separations are often troublesome and the avoidance/reduction of radioactive waste is of increasing importance (especially relevant if there are a large number of tests).
Titrimetric tests are continuous and can be carried out both with natural and synthetic substrates, but they frequently suffer from a fairly low sensitivity and are susceptible to conditions that influence the amount of protons released.
Enzymatic or chromatographic methods for the detection of one of the products of the lipolytic reaction (e.g., glycerol) are very sensitive and relatively robust, but are also complicated in terms of handling, as they demand the working-up of the incubation batch of the lipase reaction before the actual enzymatic/chromatographic detection. The coupling of an enzyme test allows only endpoint measurements (xe2x80x9ctime-stopxe2x80x9d measurement). Furthermore, in the investigation of unknown substances (e.g., searching for potential inhibitors), an effect on the enzymes of the detection reaction cannot be excluded in principle and therefore necessitates appropriate controls.
These considerations gave the impetus to the development of fluorimetric/photometric processes. In principle, these achieve the sensitivity of radiometric methods but necessitate the use of synthetic substrates or samples modified with fluorophores or chromophores. Traditional fluorimetric/photometric methods, like the radiometric procedures, are discontinuous in course and necessitate the separation of the substrate from the product. Recently, continuous fluorimetric/photometric assays have been developed (S. Hendrickson, Analyt. Biochem 219 (1994) 1-8), which are based on a shift in the fluorescence or extinction maximum of the product in comparison with the substrate. However, all these processes are restricted to the detection of phospholipases, lipoprotein lipase (LPL), cholesterol esterase, sphingomyelinase, and glucosylceramide glucosidase. Substrates having fluorophoric/chromophoric groups, suitable for the continuous activity measurement of tryglyceride-cleaving enzymes (e.g., HSL, monoglyceride lipase, diglyceride lipase, triglyceride lipase, LPL, pancreatic lipase, hepatic lipase, bacterial lipase, phospholipase A2 (PLA2), phospholipase C (PLC), cholesterol esterase), are as yet unknown.
It is therefore the aim of the invention to develop a simple continuous test for the identification of structures which favor the arrangement of aromatics to give charge-transfer complexes, such as complex phospholipid/lipid structures (bilayer, monolayer, aggregates, micelles), with the aid of synthetic fluorescence-labeled acylglycerides, and a process for determination of the activity of lipid-binding proteins, such as lipases.
Lipid transporters are proteins that recognize lipids and do not cleave like lipases, but instead transport through biological membranes.
Lipases are understood here as meaning biologically relevant endogenous lipases, such as are defined, for example, in R. D. Schmid, R. Verger, Angew. Chem. 110 (1998) 1694-1720.
A hormone-sensitive enzyme is understood as meaning an enzyme that is influenced in its activity by secondary messengers (e.g., cyclic adenosine monophosphate (cAMP)) of dependent phosphorylation or by means of other allosteric mechanisms (e.g., protein-protein interaction) which are under hormone control. Hormones that regulate the CAMP level are, for example, adrenalin, noradrenalin, glucagon, and insulin.
The invention relates to a process for the preparation of a substrate, comprising
a) reacting a fatty acid provided with a fluorescent label with 2,3-epoxypropanol to give a monoacylglyceride in alcoholic solution, such as, for example, C1-C4-alkanol, preferably methanol, at room temperature with addition of a base, such as, for example, a non nucleophilic inorganic base, preferably alkali metal carbonates and alkali metal C1-C4-alkanolates, particularly preferably methanolates, such as sodium methanolate or potassium methanolate,
b) subjecting this monoacylglyceride to ultrasonic treatment with phospholipids in the ratio (mg/ml) 1:10 to 10:1, preferably 1:2 to 3:1, and particularly preferably 1:1 to 1.5:1, from which the substrate results, which is recognizable by a color change from yellow to red.
A fluorescent label is defined as a chemical group within a molecule, which, after excitation by light, is itself capable of emitting light. Such groups are employed here in order to prepare substances which themselves are still detectable in lowest concentrations of about 1 nM. Mention may be made, for example, of N,N-dimethylaminosulfonic acid (dansyl) or 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD), preferably NBD.
A fatty acid is understood, for example, as meaning a long-chain carboxylic acid, which is saturated or unsaturated and has a chain length of C-8 to C-20, preferably C-12, C-14, C-16, and C-18 which is saturated or unsaturated, particularly preferably C-12 and saturated.
A fatty acid provided with a fluorescent label was coupled to a monoacylglyceride using 2,3-epoxypropanol. From this synthetic substrate and phospholipids, such as, for example, phosphatidylinositol and phosphatidylcholine on their own or together, optionally in a ratio by weight of 10:1 to 1:10, preferably 3:1 to 1:3, particularly preferably 2:1, micelles or vesicles which serve as a substrate of the lipase to be investigated were formed by ultrasonic treatment which lasts, for example, for about 1 to 10 minutes, preferably 1 to 6 minutes, particularly preferably 4 minutes. This incorporation into micelles or vesicles is associated with a color change from yellow to red, based on a charge-transfer complex of the aromatics, which are spatially closely adjacent in this structure. Incubation with lipase leads to the removal of the fatty acids with release of labeled fatty acid and glycerol.
As phospholipids, for example, phosphatidylcholine (6 mg) and phosphatidylinositol (6 mg) are dissolved in chloroform (1 ml each). For the preparation of the substrate, two parts of phosphatidylinositol solution (e.g., 83.5 xcexcl) and one part of phosphatidylcholine solution (e.g., 41.5 xcexcl) and 100 xcexcl of 2,3-dihydroxyprmpyl 12-(7-nitro benzo[1,2,3]oxadiazol-4-ylamino)dodecanoate (NAG) solution (10 mg in 1 ml of chloroform) were pipetted together (final concentration in the test: 0.0375 mg of phospholipid/ml; 0.05 mg/NAG/ml). After removal of the chloroform, 20 ml of 25 mM TRIS/HCl, pH 7.4; 150 mM NaCl was added and two ultrasonic treatments were carried out using an ultrasonic probe (Branson Sonifier type II, standard microtip, 25 W): 1st treatment: setting 2, 2xc3x971 min, in between 1 min each on ice; 2nd treatment: setting 4, 2xc3x971 min, in between 1 min each on ice. During this procedure, the color of the substrate solution changed from yellow (extinction maximum 481 nm) to red (extinction maximum 550 nm) due to intercalation of NAG between the phospholipid molecules of the vesicles/micelles.
The free fatty acid forms no micelles or vesicles. Therefore a color change from red to yellow was observed during the removal of the fatty acid from the micelles/vesicles. Thus, the destruction of the micelles/vesicles and the enzyme activity of the lipase is measurable, either visually (at 481 nm, or at 550 nm), by means of the color change from red to yellow, with the aid of a cuvette photometer (e.g., DU-640 from Beckman (Munich)) or of a microtiter plate reader (e.g., Microxcex2eta from Wallac (Turku, Finland)) or alternatively fluorimetrically with the aid of a phosphorimager (e.g., Storm 840 from Molecular Dynamics (Krefeld)), of a fluorescence scanner (e.g., DA-2 from Shimadzu (Osaka, Japan)), or of an image analysis process (e.g., ArrayScan from Molecular Devices (USA)) which is based on a CCD camera (charge-coupled device), which has an integrated circuit for the processing of electric and optical signals wherein the information is stored and transmitted in the form of electrical charges.
All these methods are preferably employed in combination with high-throughput screening (HTS). Further processes that are based on this concept are the measurement of the cytotoxicity of compounds and the action of detergents.
The invention also relates to a substrate prepared by the process described above and a substrate for use in a process for the identification of structures which favor the arrangement of aromatics to give charge-transfer complexes, preferably for the identification of phospholipid/lipid structures, particularly preferably of lipases/lipase inhibitors, as described above. The process can also be employed for the destruction of mono- or bilayer structures, which are curved (e.g., micelles or vesicles) or planar (e.g., artificially produced straight bilayers), which is accompanied by a color change. The color change can be monitored visually/optically or in a fluorimetrically measurable manner, as described above.
The invention further relates to a process for the preparation of the monoacylglyceride 2,3-dihydroxypropyl 12-(7-nitrobenzo[1,2,3]oxadiazol-4-ylamino)dodecanoate, where 12-aminolauric acid is first reacted with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and the intermediate obtained is then reacted with 2,3-epoxypropanol in alcoholic solution at room temperature with addition of a base, such as alkali metal carbonate and alkali metal C1-C4-alkanolate, preferably methanolate, such as sodium methanolate or potassium methanolate, and the monoacylglyceride 2,3-dihydroxypropyl 12-(7-nitrobenzo[1,2,3]oxadiazol-4-ylamino) dodecanoate itself.
The object of the invention is achieved by a process for the identification of lipases/lipase inhibitors whose presence produces a color change, comprising
a) preparing a substrate as described above,
b) incubating this substrate with a lipase (such as, for example, a HSL, monoglyceride lipase, diglyceride lipase, triglyceride lipase, LPL, pancreatic lipase, hepatic lipase, bacterial lipase, PLA2, PLC, cholesterol esterase, preferably a HSL and a pancreatic lipase, particularly preferably a HSL), and
c) determining the color change, e.g., visually/optically or fluorimetrically.
The invention further relates to lipases and lipase inhibitors that have been identified by the process described above.
The invention likewise relates to a process for determination of the activity of lipases/lipase inhibitors, where a substrate such as described above is prepared, this substrate is incubated with a lipase, and the rate of color change from red to yellow or conversely from yellow to red is determined and the activity is ascertained, for example, by means of an absorption measurement with a photometer or a fluorescence measurement with a fluorimeter.
A typical reaction is carried out at 30xc2x0 C. for 60 min, for example in 1.5 ml Eppendorf vessels or 96-hole plates. 10 xcexcl of a test substance (e.g., inhibitors of HSL) are introduced in assay buffer (25 mM TRIS/HCl, pH 7.4; 150 mM NaCl) in the presence of 16.6% DMSO. 180 xcexcl of the substrate solution (20 xcexcg/ml of phosphatidylcholine, 10 xcexcg/ml of phosphatidylinositol, 50 xcexcg/ml of NAG in assay buffer) are added. After a preincubation for 15 min at 30xc2x0 C., 20 xcexcl of HSL in assay buffer are pipetted in and the extinction is immediately measured (see above) at 481 nm in a cuvette photometer (0.5 ml cuvette) or microtiter plate reader. After a certain incubation time, which is variable and depends on the chosen enzyme concentration and can be between 2 and 240 minutes, in this case incubation at 30xc2x0 C. for 60 min, the extinction is measured again. The increase in the extinction in the yellow region, in this case at 481 nm, is a measure of the enzyme activity.
Assay systems for identification of a lipase inhibitor or for determination of the activity of a lipase/lipase inhibitor are likewise a subject of the invention. They comprise a substrate such as described above, an ultrasonic device, and optionally a device for the visual/optical and/or fluorimetric determination of the color change from red to yellow or, in addition to a substrate as described above and an ultrasonic device, a device for determination of the rate of color change and a device for absorption or fluorescence measurement.
The assay system can also be present in the form of a kit, the assay being a lipase assay.
The kit contains a substrate as described above, optionally in an assay buffer, and a container for carrying out the test, such as an Eppendorf vessel or a microtiter plate, preferably a microtiter plate.
Further subjects of the invention relate to processes for determination of molecular transport/transfer systems, for measurement of the detergent action of compounds, or for investigation of the cytotoxicity of compounds (medicaments and the like) comprising a substrate as described above and a phospholipase, for example PLA2 (from snake venom) or PLC (Bacillus cereus).
Transporters/transfer proteins are understood as meaning proteins which themselves recognize principal nutrients such as carbohydrates, lipids, and proteins and transport them through biological membranes or transfer them from a certain biological membrane to another. Transporters/transfer proteins, for example isolated from rat ileum, are functionally reconstituted (proteoliposomes) in phospholipid vesicles (liposomes) or incubated together with liposomes as soluble polypeptides and then added to a mixture of phospholipids and NBD-glyceride as described above and treated with ultrasound. The transport process or transfer process of the NBD-glyceride from the NBD-glyceride-containing micelles/vesicles into the lumen of the proteoliposomes with the aid of the transporters or into the membrane of the liposomes with the aid of the transfer proteins leads to the dissolution/destruction of the micelle structure and can in turn be monitored photometrically or fluorimetrically as described above.
The detergent action of chemical compounds is based on the direct destruction of the micelles/vesicles. As biological membranes are also constructed in this way, such compounds are usually cytotoxic. Destruction of micelles can easily be detected using the present process by means of the described color change.
Syntheses:
A few NBD-labeled fatty acids such as 12-(7-nitrobenzo[1,2,3]oxadiazol-4-ylamino)dodecanoic acid(1) are indeed commercially available, but expensive. Although the first experiments were also carried out with commercially obtainable material, it was possible to obtain compound (1) in good yields by reaction of 12-aminolauric acid with 4-chloro-7-nitrobenzo[1,2,5]oxadiazole in MeOH.
By nucleophilic addition of (1) to 2,3-epoxypropanol (2), it was possible to obtain the monoacylglyceride (3) in good yields. Compound (3) was then acylated in order to reach the triglycerides (4) and (5). The diacylglyceride (6) was obtained by esterification of (1) with palmitin. This compound was also reacted to give the corresponding triacylglyceride (7). The same method was used in order to react the glycerol diether (8) to give the pseudotriacylglyceride (9), wherein two acyl radicals are replaced by long-chain ethers.
All compounds synthesized proved to be substrates of lipases, preferably of HSL, but showed considerable activity differences. The xe2x80x9cbestxe2x80x9d substrate of the lipases proved to be the monoacylglyceride (3). The introduction of further acyl groups, as in the compounds (4), (5), (6), and (7), led to a decrease in activity.
This can easily be explained by competition in the removal of the NBD acyl group by the newly introduced acyl radicals. In order to confirm this hypothesis, a pseudotriacylglyceride (9) was synthesized wherein two acyl groups are replaced by hexadecyl ether units. These cannot be removed from the lipases and should therefore not compete with the NBD fatty acid ester. In biological tests, this compound indeed proved to be a substrate, but with low activity. Obviously, in addition to the catalytic region, the lipases have an extended hydrophobic binding region accessible to the long-chain ether groups, such that the addition of the fatty acid unit is impeded.
As the monoacylglyceride (3) proved to be a good substrate of the lipases, it was investigated whether there is a regioselective preference for position 1 or 3. For these investigations, the enantiomeric regioisomers (3a) and (3b) were synthesized.
The synthesis of the enantiomers (3a, b) starts from D- and L-1,2-O-isopropylideneglycerol, which is esterified with the NBD-labeled fatty acid (1) by dicyclohexylcarbodiimide (DCC) activation. The protective group was removed using 1 N methanolic HCl. Both compounds showed identical biological activity, such that the use of enantiomerically pure compound promises no advantage.